Summary of work: GADD153 is a highly conserved mammalian gene whose expression is increased in response to a variety of stresses including those associated with growth arrest and DNA damage. It has been implicated in both the induction of growth arrest and apoptosis, but its functional role in these processes is still far from clear. GADD153 is a member of the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors. It cannot bind to consensus C/EBP binding sites, but can act as a negative regulator of these C/EBPs by virtue of its ability to dimerize with them and inhibit their binding to DNA. It has also recently been suggested that GADD153-C/EBPbeta dimers can act as positive regulators of transcription through interaction with novel sites. Studies in this project have focused on the regulation of GADD153 gene expression during stress and the function of the GADD153 protein during the cellular response to diverse stimuli. With respect to regulation, efforts over the past year have concentrated on the role of two CREB-family transcripiton factors in regulating GADD153 transcription during stress. We have provided evidence that CREB2 acts as a transcriptional activator, while ATF3 acts as a repressor of GADD153 expression following sodium arsenite treatment. Acting in a sequential manner these transcription factors bind to the C/EBP site in the GADD153 promoter and contribute to the biphasic induction of GADD153, emphasizing the importance of both positive and negative regulatory factors in controlling GADD153 expression during stress. Studies on the function of GADD153 have focused on its influence on cell survival during stress. Employing a model system in which GADD153 protein is constitutively expressed at high levels in myc-transformed Rat1 cells, we have found that GADD153 overexpression leads to enhanced sensitivity of a variety of cells to stressful treatments. This is correlated with reduced levels of Bcl-2 protein and current studies are exploring the mechanism whereby GADD153 represses Bcl-2 expression. Our findings are consistent with GADD153 serving a pro-apoptotic function.